Bifunctional chelating agent

ABSTRACT

The invention concerns a ligand comprising wherein n is an integer from 1 to 5, X represents —NO 2 , —NH 2 , —NCS, —NHCOCH 2 -Z. NHCO—W—COCNHS, —NH-Q, —NHCS-Q, —NHCOCH 2 -Q, or —NHCO(CH 2 ) m ?-Q where Q is an hapten chosen from the group consisting of steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof or any activated linker ready for coupling reaction, Y is CO 2 H or PO 3 H 2  W is —(CH 2 ) m — m is an integer from 1 to 10. Z is chloride, bromide or iodine

TECHNICAL FIELD

The present invention relates to bifunctional chelating agents and moreparticular to bifunctional polyaza polycarboxylic or polyphosphonicmacrocycles ligands a method of synthesis of these products and theiruses.

BACKGROUND OF THE INVENTION

Alpha-emitting radionuclides are good potential candidates forradioimmunotherapy. ²²⁵Ac decays through a chain of 3 alpha emissions to²¹³Bi.

As described by Davis, I. A. et al in the article “Comparison ofActinium 225 Chelates: Tissue Distribution and Radiotoxicity” publishedin Nucl. Med. Biol., Vol. 26, pp 581–589, 1999; by Deal, K. A. et al. inthe article “Improved in Vivo stability of Actinium-225 MacrocyclicComplexes” published in J. Med. Chem., Vol. 42, pp. 2988–2992, 1999 andby Grote Gansey, M. H. B. et al. in the article “Conjugation,Immunoreactivity, and Immunogenicity of Calix (4) arenes; Model Study toPotential Calix (4) arenes—Based Ac3+Chelators” published in Bioconj.Chem., Vol.10, pp 610–623, 1999, the stability of bifunctional chelatingagent of actinides, lanthanides and bismuth is not satisfactory.

An object of the invention is to provide more effective bifunctionalchelating agents for metals especially actinides and lanthanides andbismuth.

SUMMARY OF THE INVENTION

The present invention includes a ligand comprising:

wherein

-   -   n is an integer from 1 to 5    -   X represents —NO₂, —NH₂, —NCS, —NHCOCH₂-Z, NHCO—W—COCNHS, —NH-Q,        —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q where Q is an hapten        chosen from the group consisting of steroids, enzymes, proteins,        monoclonal antibodies, chimeric antibodies, or fragments thereof        or any activated linker ready for coupling reaction,    -   Y is CO₂H or PO₃H₂    -   W is —(CH₂)_(m)—    -   m is an integer from 1 to 10    -   Z is chloride, bromide or iodine.

If X represents —NH-Q, —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q where Qis a hapten chosen from the group consisting of steroids, enzymes,proteins, monoclonal antibodies, chimeric antibodies, humanisedantibodies or fragments thereof, the resulting ligand is also called aligand-hapten conjugate.

The invention also includes according to a preferred embodiment, a metalchelate of the ligand as described above wherein the metal is chosenfrom the group consisting of the lanthanides and the actinides. Themetal is preferably actinium and most preferably actinium-225 (²²⁵Ac).

The invention also includes according to a preferred embodiment, a metalchelate of the ligand as described above wherein the metal is preferablybismuth and most preferably bismuth-213 (²¹³Bi).

The present invention also includes a process for the preparation ofsaid ligand, said process comprising a bimolecular cyclization betweenan iminodiester and a polyamine by the action of a molar equivalent ofsodium methoxide.

The present invention also includes the method of using the metalchelates of the ligand-hapten conjugate possessing a linking groupwherein the chelate as a therapeutic or diagnostic agent.

Specifically, such ligands are useful for radiolabeling proteins withradioactive metals, and can consequently be utilised with respect toradioimmunoimaging and/or radioimmunotherapy. The present theligand-hapten conjugates firmly link metals especially actinides,lanthanides and bismuth to proteins, minimise metal release and permithigh selective delivery of metals to targeted sites in vivo. This isespecially true for the actinium and bismuth complexation metal chelateprotein conjugates.

Immunotherapy with radiolabelled antibodies allows fairly specifictargeting of certain cancers (see f.ex. Couturier, O. et al. “Validationof 213-Bi-alpha radioimmunotherapy for multiple myeloma” in Clin.Cancer. Res., Vol. 5, pp. 3165–3170, 1999; Huneke, R. B., et al. in“Effective alpha-particle-mediated radioimmunotherapy of murineleukemia” in Cancer Res., Vol. 52, pp. 5818–5820, 1992; Kennel, S. J. etal. “Radioimmunotherapy of micrometastases in lung with vasculartargeted 213Bi” in Br. J . Cancer, Vol. 80, pp. 175–184, 1999; Kozak, R.W. et al. “Bismuth-212-labeled anti-Tac monoclonal antibodyalpha-particle-emitting radionuclides as modalities forradioimmunotherapy” in Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 474–478,1986 or Macklis, R. M. et al. “Radioimmunotherapy withalpha-particle-emitting immunoconjugates” in Science, Vol. 240, pp.1024–1026, 1988.

This technique is based on the use of radionuclides associated toantibodies or peptides that are specific of antigens expressed on thetumour cells. In order to bind a radionuclide to a vector it isnecessary to use bifunctional chelating agents (BCA) that have twospecific sites. One site is to be coupled to the vector and the otherhas to form very stable complexes with the radionuclide to be used.

²²⁵Ac and ²¹³Bi are good candidates for such applications as describedby Kaspersen, F. M. et al. “Cytotoxicity of 213Bi- and225Ac-immunoconjugates” in Nucl. Med. Commun., Vol. 16, pp. 468–476,1995. The very short range (<100 μm) of α-particles and the high energytransfer allows efficient destruction of tumor cells whereas normalcells are relatively spared.

Chelators that can hold radioactive metals with high stability underphysiological conditions are essential to avoid excessive radiationdamage to non-target cells.

Furthermore, these bifunctional chelating agents allow differentapplications; it can be used to bind ²²⁵Ac or other actinides andlanthanides or ²¹³Bi to any biological or non-biological structures forany applications.

These chelating agents can be used non-associated to a vector as adetoxication chelating agent or using the natural tropism of thecomplex.

This chelating agent can also be used grafted on a chromatographiccolumn in order to purify or concentrate any solutions containing ²²⁵Acor other actinides, lanthanides or ²¹³Bi.

The complexation properties of our product with ²²⁵Ac or other actinidesor lanthanides show that this chelating agent may also be useful as agood extractant in the process of separation of minor actinides andlanthanides in nuclear waste or to separate specific groups of metals inhigh level waste.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention are described by way of example andwith reference to the accompanying drawing wherein:

An access route that allows the synthesis of a bifunctionalmacrocycle-chelating agent is described in FIG. 1.

FIG. 1. represents a scheme for the preparation of a substituted1,4,7,10,13,16-hexa(2-carboxymethyl)-hexaazacyclooctadecane ligand(HEHA).

DETAILED DESCRIPTION

Different non-functionalised chelating agents (commercially available orreadily synthesised in the laboratory) bearing aminocarboxylate groups(EDTA, DTPA, DOTA, PEPA, and HEHA) or aminophosphonate groups (EDTMP)were tested for their complexation properties with ²²⁵Ac and ²¹³Bi. Itwas found that HEHA compound(1,4,7,10,13,16-hexacarboxylmethyl-1,4,7,10,13,16-hexaazacyclooctadecane)appeared to be the best candidate for ²²⁵Ac complexation. This result isin balance in regard of previous studies. Polyaza polycarboxylicmacrocycles are known to form thermodynamically stable complexes withlarge metal ions such as actinides and lanthanides.

It was also found that HEHA appeared to be a good chelating agent of²¹³Bi.

Therefore, after selection of the suitable candidate, a method of thesynthesis of the C-functionalised analogue was set up. To achieve that,the2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexakis(2-carboxymethyl)-hexaazacyclooctadecanecompound which is functionalised at C-2 on the cycle by either anisothiocyanate termination for future covalent attachment tobiomolecules or any activated linker ready for coupling reaction wasprepared.

Macrocyclic polyamines, the key precursors to macrocyclic bifunctionalchelating agents are synthesised by different way: the Richman-Atkinscyclization of deprotonated tosylamides with tosylates in aproticsolvents, the <<crablike<< (cyclization of a bis (α-chloroamide) withamines, the Tabushi cyclization (aminolyse of malonates with polyamines)or via peptide synthesis and intramolecular tosylamide ring closure.

However, the efforts to prepare the2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexakis(2-carboxymethyl)-hexaazacyclooctadecanewith these classical methods failed.

A different synthetic route to the bifunctional macrocycles viaaminolyse of an iminodiester with a polyamine of in the presence ofNaOMe was developed. The reaction betweenN-methoxycarbonylmethyl-p-nitrophenylalanine methyl ester andtetraethylenepentamine upon refluxing in methanol for several days inthe absence of sodium methoxide were not concluent.

An improved procedure for the bimolecular cyclization between animinodiester and a polyamine by the action of a molar equivalent ofsodium methoxide is described in more detail. Yield of 50% was obtainedto prepare the bifunctional dioxoaza macrocycle without resorting tohigh dilution. This surprising methodology which was developed is simpleand convenient and allows preparation of functionnalised macrocyclicpolyamines of varying ring size.

Referring now to the FIGURE, the synthesis of2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexa(2-carboxymethyl)-hexaazacyclooctadecaneis described in more detail.

The commercial product, 4-nitrophenylalanine (1) was used as startingmaterial.

Treatment of (1) with HCl gas in methanol led to the4-nitrophenylalanine methyl ester hydrochloride (2). Compound (2) wasmonoalkylated by methylbromoacetate in the presence of triethylamine togive diester N-((methoxycarbonyl)methyl)-4-nitrophenylalanine methylester (3). Treatment of (3) with tetraethylenepentamine in the presenceof sodium methanolate in refluxing methanol resulted in macrocyclisationto give the cyclic diamide3-(4-nitrobenzyl)-2,6-dioxo-1,4,7,10,13,16-hexaazacyclooctadecane (4).Reduction with BH₃ afforded after treatment with HCl gas andpurification by anion-exchange chromatography the2-(4-nitrobenzyl)-1,4,7,10,13,16-hexaazacyclooctadecane (5). Treatmentof (5) with ter-butyl bromoacetate in the presence of sodium carbonategave the hexaester2-(4-nitrobenzyl)-1,4,7,10,13,16-hexakis-(tert-butoxycarbonylmethyl)-1,4,7,10,13,16-hexaazacyclooctadecane(6). The nitrobenzyl function was selectively reduced by using tinchloride in ethanol to obtain the aminobenzyl compound2-(4-aminobenzyl)-1,4,7,10,13,16-hexakis(tert-butoxycarbonylmethyl)-1,4,7,10,13,16-hexaazacyclooctadecane(7). Cleavage of the ester groups with trifluoroacetic acid followed bypurification on ion exchange chromatography column and treatment withthiophosgene gave the final compound2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexa(2-carboxymethyl)-hexaazacyclooctadecane (8) (AOI032).

An alternative to isothiocyanato coupling function can be used byintroducing different activated linkers. It is also possible to replaceaminocarboxylate groups by aminophosphonate groups to complex the metalto be used.

In conclusion, different non-functionalised chelating agents bearingaminocarboxylate or aminophosphonate groups were tested for theircomplexation properties with ²²⁵Ac and ²¹³Bi. After selection of thebest candidate, the C-functionalised analogue was synthesised.

EXAMPLE 4-Nitrophenylalanine methyl ester hydrochloride (2)

4-Nitrophenylalanine (1) (24 mmol) was treated with methanol (100 ml)saturated with HCl (g) and left to stir at room temperature for 18hours. The solution was concentrated by evaporating to ⅓ of originalvolume and the precipitate was collected and dried under vacuum for 18hours. The yield was 85%.

NMR ¹H (250 MHz, D2O): δ 8.2 (d, 2H), 7.53 (d, 2H), 4.55 (t, 1H), 3.84(s, 3H), 3.43 (m, 2H).

N-((Methoxycarbonyl)methyl)-4-Nitrophenylalanine methyl ester (3)

Triethylamine (22 mmol) was added to a suspension of4-Nitrophenylalanine methyl ester hydrochloride (2) (21 mmol) in THF (50ml). The mixture was stirred at room temperature for one hour, thetriethylamine hydrochloride was filtered off, and the filtrateconcentrated to yellow oil. The oil was dissolved in dry THF (50 ml) andto this solution was added triethylamine (60 mmol) andmethylbromoacetate (60 mmol), the solution was stirred at roomtemperature under nitrogen atmosphere for 3 hours, after which theprecipitate was filtered off and the filtrate concentrated on vacuum.The residue was dissolved in ethylacetate, washed with H₂O, dried(MgSO₄) and concentrated on vacuum to give yellow oil. The yield was92%.

MS (M+1): 297

NMR ¹H (250 MHz, CHCL3): δ 8.2 (d, 2H), 7.53 (d, 2H), 3.7 (m, 7H), 3.3(m, 2H), 3.1 (m, 2H).

3-(4-nitrobenzyl)-2,6-dioxo-1,4,7,10,13,16-hexaazacyclooctadecane (4)

Sodium (20 mmol) was dissolved in dry methanol (100 ml) at roomtemperature under nitrogen atmosphere and to this solution was addedtetraethylenepentamine (18 mmol) andN-((Methoxycarbonyl)methyl)-4-Nitrophenylaianine methyl ester (3) (18mmol). This solution was refluxed for 72 hours after which the solventwas removed and the residue was purified on silica gel chromatographywith chloroformelmethanol/NH₃ (aq) (75:20:5), affording a yellow powder.The yield was 50%.

MS (M+1): 422

IR (Kr, cm⁻¹); 3287 (NH); 3287–2842 (Ar—C—H); 1656 (C═O); 1517 and 1345(NO₂)

NMR ¹H (250 MHz, CDCL₃): δ 8.17 (d, 2H), 7.57 (s, NH amide), 7.40 (d,2H), 7.27 (s, NH amide), 3.14–3.48 (m, 9H), 2.6–2.9 (m, 11H).

NMR ¹³C (CDCI₃): CO: 175, 145, 130, 123, 55, 52, 40

2-(4-nitrobenzyl)-1,4,7,10,13,16-hexaazacyclooctadecane (5)

A solution of BH₃ in THF (100 mmol) was added dropwise to a stirredsuspension of3-(4-nitrobenzyl)-2,6-dioxo-1,4,7,10,13,16-hexaazacyclooctadecane (4)(10 mmol) in THF (50 ml) at 0° C. under nitrogen atmosphere. Thesolution was heated at reflux for 36 hours. Methanol was added slowly tothe solution at 0° C. after which the solvent was removed and theresidue was dissolved in methanol (50 ml); the resulting mixture wascooled at 0° C. and gaseous HCl was bubbled through the solution andthen the mixture was refluxed for 12 hours. The resulting precipitatewas collected washed with ether to give a white powder. The solid wasdissolved in water and was loaded on a column of DOWEX 1X-8anion-exchange resin (OH⁻ form). The column was eluted with water;alkaline fractions were collected, and the water was removed undervacuum to give pale yellow oil. The yield was 55%.

MS (M+1): 394

IR (Kr, cm⁻¹): 3428 (NH); 2961–2759 (Ar—C—H), 1518 and 1349 (NO₂)

The I.R. spectrum showed no band at 1656 cm⁻¹ for the C═O group.

NMR ¹H (250 MHz, CDCL₃): δ8.06 (d, 2H), 7.27 (d, 2H), 2.3–2.9 (m, 25H)

2-(4-nitrobenzyl)-1,4,7,10,13,16-hexakis(tert-butoxycarbonylmethyl)-1,4,7,10,13,16-hexaazacyclooctadecane (6)

To a solution of 2-(4-nitrobenzyl)-1,4,7,10,13,16-hexaazacyclooctadecane(5) (10 mmol) in DMF (50 ml) at room temperature under a nitrogenatmosphere was added anhydrous sodium carbonate (0.11 mol) and asolution of tert-butyl bromoacetate (62 mmol) in DMF (20 ml). Themixture was heated at 60° C. for 18 hours after which the solvent wasremoved and the residue was dissolved in chloroform washed with brine,dried (MgSO₄) and concentrated on vacuum to give yellow oil. The yieldwas 82%.

MS (M+1): 1078

2-(4-aminobenzyl)-1,4,7,10,13,16-hexakis(tert-butoxvcarbonvlmethyl)-1,4,7,10,13,16-hexaazacyclooctadecane (7)

To a solution of2-(4-nitrobenzyl)-1,4,7,10,13,16-hexakis(tert-butoxycarbonylmethyl)-1,4,7,10,13,16-hexaazacycloocta (6) (15 mmol) in ethanol (50 ml) atroom temperature under a nitrogen atmosphere was added SnCl₂ (0.125mol). The mixture was refluxed for 12 hours after which the solvent wasremoved and the compound thus obtained was dissolved in water; thesolution was brought to pH 8 with 2M NaOH. The resulting precipitate wasremoved off and the filtrate concentrated on vacuum; the residue wasdissolved in acetonitrile and passed over Celite. The filtrate wasevaporated on vacuum to give yellow oil. The yield was 62%.

MS (M+1): 1048

2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexakis(2-carboxymethyl)-1,4,7,10,13,16-hexaazacyclooctadecane (8)

2-(4-aminobenzyl)-1,4,7,10,13,16-hexakis(tert-butoxycarbonylmethyl)-1,4,7,10,13,16-hexaazacyclooctadecane (7) (10 mmol) was treated withTFA (0,1 mol) 6 hours at room temperature under nitrogen atmosphereafter which the solvent was removed. The compound thus obtained wasdissolved in water and loaded on a column of DOWEX 50WX8-200 (H⁺ form).

The column was eluted consecutively with 0.5 M HCl and with water untilthe eluant was neutral and finally with 0.5 M aqueous ammonia solution.Alkaline fractions were collected, and the water was removed on vacuumto give 2-(4-aminobenzyl)-1,4,7,10,13,16-hexakis(2-carboxymethyl)-1,4,7,10,13,16-hexaazacyclooctadecane as pale yellow oil.

MS (M+1): 712

NMR ¹H (250 MHz, D₂O): δ 7.08 (d, 2H), 6.80 (d, 2H), 2.5–4.0 (m, 37H)

The compound thus obtained was dissolved in water and the pH wasadjusted to 9.0 with NaHCO₃. To this solution was added at roomtemperature under nitrogen atmosphere thiophosgene in CHCl₃ (10 ml), themixture was stirred for 2 hours. The organic layer was removed and thewater was evaporated on vacuum to give the final product (8) (AOl032).

The yield was 65%.

MS (M+1): 754

The I.R. spectrum showed a strong band at 2100 cm for the aryl SCNgroup.

While a preferred embodiment of the present invention has beendescribed, it will apparent to those skilled in the art that manychanges and modifications may be made without departing from theinvention in its broader aspects. The appended claims are thereforeintended to cover all such changes and modifications as fall within thetrue spirit and scope of the invention.

1. A ligand comprising

wherein n is an integer from 2 to 5 X represents —NHCO—(CH2)m-COCNHS,—NH-Q, —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q and where Q is an haptenchosen from the group consisting of steroids, enzymes, proteins,monoclonal antibodies, chimeric antibodies; and m is an integer from 1to
 10. 2. A metal chelate of the ligand of claim 1, wherein the metal ischosen from the group consisting of the lanthanides and the actinides.3. A metal chelate of the ligand of claim 2, wherein the metal isactinium.
 4. A metal chelate of the ligand of claim 3, wherein the metalis actinium-225 (²²⁵ Ac).
 5. A metal chelate of the ligand of claim 1,wherein the metal is bismuth.
 6. A metal chelate of the ligand of claim5, wherein the metal is bismuth-213.
 7. A metal chelate of a ligandcomprising

wherein n is an integer from 2 to 5 X represents —NHCO—(CH2)m-COCNHS—NH-Q, —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q and where Q is an haptenchosen from the group consisting of steroids, enzymes; proteins,monoclonal antibodies, chimeric antibodies m is an integer from 1 to 10wherein the metal is actinium, and wherein said ligand is prepared by aprocess comprising a bimolecular cyclization between an iminodiester anda polyamine by the action of a molar equivalent of sodium methoxide. 8.A metal chelate of the ligand of claim 7, wherein the metal isactinium-225 (²²⁵Ac).